Melting curve analysis for detecting nucleic acid sequence variation is to include an additional temperature increasing step (sometimes may also be a temperature decreasing step) in the real-time PCR program, then, information about amplification products or sequence variations can be detected by recording the change of fluorescence with the change of temperature. The current melting temperature analysis includes three types, namely the fluorescent dye method, the fluorescent probe-based method, and the fluorescent dye-fluorescent probe combination method.
The principle of the fluorescent dye method is very simple, wherein a dye (such as SYBR Green, SYTO-9, or LC Green) capable to bind double-stranded DNA molecules to give rise to fluorescence is added into the PCR system. An increase in temperature leads to denaturation of the double-stranded DNA, resulting in a decrease of fluorescence. A sequence variation can be indicated as a change in melting temperature (Wittwer C. T., et al, BioTechniques, 1997, 22:130-138; Ririe K. M., et al, Anal. Biochem, 1997, 245:154-160; US patent US 2006/0019253 A1); US patent, US 2003/0224434 A1). Specifically, single nucleotide changes can be detected in combination with High Resolution Melting (HRM) analysis (Wittwer C. T., et al, Clin Chem, 2003, 49: 853-860). Fluorescent probe-based method is using probes to detect sequence variations at a specific site, provided that the probe can give rise to a specific fluorescence signal upon hybridizing to a target sequence. There are various types of such probes in real-time PCR, but few probes are available for melting curve analysis, among them, the most well-known is the Fluorescence Resonance Energy Transfer (FRET) probe, also called LightCycler™ probe or adjacent hybridization probe (U.S. Pat. No. 7,160,998 B2; U.S. Pat. No. 6,472,156 B1; U.S. Pat. No. 6,140,054). Others use oligonucleotide probes with a single label (U.S. Pat. No. 6,635,427 B2), HyBeacon (US patent, US 2008/0311579 A1) probe, etc. A fluorescent dye and fluorescent probe in combination method is a method wherein either a fluorescence-enhancing or a fluorescence-quenching dye is added simultaneously with a fluorescent probe, such as in the so-called induced fluorescence resonance energy transfer (iFRET) technology (U.S. Pat. No. 7,179,589 B2), wherein a fluorescent intercalating dye is added simultaneously with a single-labeled fluorescent probe. In such a case, the fluorescent intercalating dye binds to double-stranded DNA and the fluorescence emitted can increase the fluorescence of the fluorescently labeled probe by energy transfer. An increase in temperature makes the probe dissociate from the target sequence, thereby decreasing the hypersensitive fluorescence. Gupta et al. (US patent, US 2007/0020665 A1) disclosed a way for molecular subtyping of the hepatitis C virus, wherein a fluorescence quenching dye and a fluorescently labeled probe are added to the PCR reaction simultaneously. The fluorescence is quenched upon hybridization, an increase in temperature makes the probe dissociate from the target sequence, thereby allowing the quenched fluorescence to recover and resulting in the fluorescence increase.
Among the three types of melting curves discussed above, the dye method employs a single fluorescence channel for detection, and it is presently mainly used for the identification of amplification products. In combination with HRM, it is used for detection of random mutations in the amplified sequence rather than for detection of mutations at a specific site. Even rarer is it used for detection of multiple mutations occurring at multiple specific sites. The fluorescent dye and fluorescent probe in combination method (regardless whether this combination involves fluorescence enhancing format or fluorescence quenching format) is limited to certain special fluorescent dyes, and the number of fluorescence channels that can be used for detection of this format of labeling is limited, so as to the number of sites that can be detected. Thus, few application examples involving this dye-probe combination method exist.
The most successful example of the probe-based method is LightCycler™ probe. LightCycler™ probe consists of two specific probes that are complementary to an adjacent region of the template. One probe is labeled with a donor fluorophore (referred to as detection probe) and the other is labeled with an acceptor fluorophore (referred to as anchor probe). The melting temperature of the detection probe is approximately 10° C. lower than that of the anchor probe. FRET should take place between the donor fluorophore and the acceptor fluorophore. In the absence of a target sequence, the two probes are separated and stay in a free state, and the acceptor fluorophore group cannot be excited, thereby generating no FRET signal. In the presence of a complementary target sequence, the two probes bind to the complementary template simultaneously, which brings the donor fluorophore group and the acceptor fluorophore group close to each other. The fluorescence energy generated by the donor fluorophore group is absorbed by the acceptor group, resulting in a fluorescence signal of a specific wavelength, and a FRET signal becomes detectable. When the temperature increases, the detection probe dissociates from the template first, and a specific melting temperature could be detected. When a sequence variation exists in target sequence that is hybridized with the detection probe, the degree of variation will affect the temperature of probe dissociation, resulting in a different melting temperature. Based on this, whether and in which specific form a sequence variation occurs in the detection probe-covered regions of the target could be determined. Since the LightCycler™ technology requires an anchor probe that is actually not used for detecting sequence variations, the area covered by the anchor probe will become a blind area for detection. When sequence variation exists in a wide range, the selection of a conserved region for the anchor probe would become difficult. In addition, since LightCycler™ probe employs the detection of FRET, a suitable wavelength combination of fluorescence donor and acceptor would be required for FRET to take place. However, only limited combinations of fluorescence donor and acceptor are currently available for carrying out effective FRET. Meanwhile, the optical channel for the detection of FRET is different from that for detection of conventional, single fluorescent dyes: with the exception of the dedicated instruments, most mainstream real-time PCR machines can not be used for the detection of FRET. Moreover, the number of channels useful for detecting FRET is limited as well, making the FRET technology greatly restrained in the application of detecting multiple genes in a single tube.
In the probe-based method, both the single-labeled oligonucleotide probe and the HyBeacon probe are oligonucleotide probes labeled only with a fluorescent group, and change of fluorescence intensity would occur after hybridization of the probe to a target. Both probes are useful in melting curve analysis, whereby nucleic acid sequence variation is detected via changes in of the melting temperature. However, in this fluorophore only single-labeling manner, no quencher group exists and quenching efficiency of the probes depends on specific nucleic acid sequence or the guanine residue. That makes the fluorescence background relatively high, changes in fluorescence intensity after hybridization being limited, and the signal to noise ratio is low. Moreover, in the case of the HyBeacon probe, the fluorescent group is labeled internally, making it difficult to synthesize and label the probes, thereby restricting wide application of such probes in the detection of nucleic acid sequence variation by melting curve analysis.
In the probe-based method, there is also a type of dual-labeled probes containing minor groove binder (MGB), especially probes with the MGB located in the 5′ end, such as MGB-Eclipse probe (Afonina, I. A., et al, Biotechniques, 2002, 32:940-944, 946-949) and Pleiades probe (Lukhtanov, E. A., et al, Nucleic Acids Res, 2007, 35: e30). Since such probes can resist the 5′-hydrolysis activity of thermostable DNA polymerase (e.g., Taq polymerase), they have also been reported to be useful in melting curve analysis. MGB group in this kind of probes may act to increase the melting temperature. The aim of this design is to shorten the probe while maintaining a relatively high melting temperature, but for a mismatched target sequence, the melting temperature will decrease a lot. Thus, it is mainly used to specifically detect the matched target sequence, rather than being used for a melting curve analysis for mutation detection. This is because the latter requires that both the matched and mutated target sequences to be differentiated through different melting temperatures, and it is not necessary that the mismatched target sequence has a very low melting temperature. In addition, the synthesis of this kind of probes is more difficult and more expensive than the synthesis of probes without MGB.
Thus, a novel fluorescent probe is needed for melting curve analysis in order to achieve simultaneous detection of multiple variations in a single tube. Preferably, such a fluorescent probe can be labeled with common fluorescent groups, and can perform multi-color analysis in a commonly used real-time PCR machine. Such a probe is also preferably suitable for the melting curve analysis of nucleic acid amplification products, for example, it will not be degraded or is only degraded minimally under the conventional PCR-cycle reaction conditions, in order to maintain sufficient amount of intact fluorescent probes for subsequent melting curve analysis. More preferably, such a probe shall be easily synthesized, not involving complicated and expensive chemical modifications, thereby lowering the cost for use.